Zero Power Visible Colorimetric Pathogen Sensors

ABSTRACT

A visibly perceived colorimetric pathogen sensor ( 100 ) can comprise a substrate ( 110 ) and an molecular recognition group ( 120 ) coupled to the substrate ( 110 ). The molecular recognition group ( 120 ) can be operable to bind to a target pathogen ( 130 ). Upon the molecular recognition group ( 120 ) binding with the target pathogen ( 130 ), reflected light can be altered thereby changing apparent color.

RELATED APPLICATION

This application claims priority to U.S. Provisional Application No. 62/926,376 filed Oct. 25, 2019 and U.S. Provisional Application No. 63/021,605 filed May 7, 2020, which are each incorporated herein by reference.

GOVERNMENT INTEREST

This invention was made with government support under grant no. 1931100 awarded by the National Science Foundation. The government has certain rights in the invention.

BACKGROUND

Detection of pathogens such as viruses can provide critical information in diagnosing and treating infected individuals. However, current methods and devices used to detect pathogens tend to be slow, inconvenient, and can sometimes be unreliable. Many methods of virus detection can involve nucleic acid and protein sequencing, immunofluorescence assays, viral plaque assays, and other methods which can require expensive equipment and expert training to perform.

For example, Sars-CoV-2 (CV2) is sensed and detected using several different techniques: a) through identification of its genetic materials (RNA), b) through detection of viral proteins (antigens) expressed by CV2 in the respiratory tract of a person, and through surface proteins. CV2 can also be indirectly detected through biomolecules (biomarkers) that are produced by the body in response to CV2 (e.g. C-reactive protein and serum amyloid). After 7-14 days of infection, the body also produces antibodies that can be detected to infer the infection. Many research laboratories and biotechnology companies use techniques such as real-time reverse transcriptase (RT) polymerase chain reaction (PCR), isothermal nucleic acid amplification of the RdRp viral target, RT-PCR with LOD of CV2 viral RNA, and the like. In these tests the amplification of RNA or the viral target is followed by a detection using fluorescent molecules that selectively bind with the target molecules. The amplification step requires time. Serological lateral flow assay (LFA) point-of-care rapid tests are also developed for antibody (IgG/IgM) detection. Serological enzyme linked immune-absorbent (ELISA) assay, microarrays, and many other detection techniques are also under development for CV2. Biomarkers and antibodies can be detected using ELISA, microarrays with conjugate molecules, and fluorescent readout.

Other virus detection methods may include direct counting (e.g. flow cytometry, TEM, etc) which are time and labor intensive, as well as requiring expensive equipment and training. As such, improvements in pathogen detection continue to be sought. Colorimetric

Colorimetric techniques based on fluorescent molecules or Förster resonance energy transfer (FRET) are extensively developed and constitute an active area of research and development. All these techniques require elaborate microscopes and optical detection systems to detect the changes in the colorimetric sensors.

SUMMARY

The main idea explored in the present patent application is to use relatively large structures (>500 nm) and components such as microbeads, flexure beams, or passive arrayed structures with enhanced mechanical, sensitivities to electrical, magnetic and optical loading by viral particles to enable optically unassisted and visible detection of the viral particles.

A colorimetric pathogen sensor is disclosed that can comprise a substrate and a molecular recognition group coupled to the substrate. The molecular recognition group can be operable to bind to a target pathogen. Upon the molecular recognition group binding with the target pathogen, reflected light can be altered thereby changing apparent color.

In one example, a colorimetric pathogen sensor is disclosed that can comprise a plurality of bases and a molecular recognition group bonded to the plurality of bases. The molecular recognition group can be operable to specifically bind to a target pathogen. Upon the molecular recognition group binding with the target pathogen, the plurality of bases can group together via the bonds to the molecular recognition group, thereby altering reflected light and changing apparent color.

In another aspect, a colorimetric pathogen sensor is disclosed that can comprise a matrix and a molecular recognition group embedded in the matrix. The molecular recognition group can be operable to bind to a target pathogen. The colorimetric pathogen sensor can also comprise a plurality of particles embedded in the matrix. Upon the molecular recognition group binding with the target pathogen, the matrix can expand and change the position of the plurality of particles relative to one another, thereby altering reflected light and changing apparent color.

In yet another aspect, a colorimetric pathogen sensor is disclosed that can comprise first and second transparent plates. Each transparent plate can have a dispersive surface. The colorimetric pathogen sensor can also comprise a matrix associated with the first and second plates. In addition, the colorimetric pathogen sensor can comprise a molecular recognition group embedded in the matrix, the molecular recognition group being operable to bind to a target pathogen. Upon the molecular recognition group binding with the target pathogen, a refractive index of the matrix can change, thereby altering reflected light from the dispersive surfaces and changing apparent color.

Thus, these devices display optical changes that occur in the sensors which are visible to the unassisted eye, such that microscopes and optical detectors are not needed. Further, the biosensors disclosed here are zero power devices and do not require batteries or other power sources. As a result, the binding between the viral particles and the sensor structure produces stresses and stiction forces that cause the deformation and deflection of the sensors components or stiction of fluorescent or colored microbeads, or enhanced response of arrayed optical structures etc. leading to a visible color change.

In another example, a method of detecting a virus can include exposing a sensor surface to a fluid sample containing a suspected virus. The sensor surface can then be irradiated with electromagnetic waves at multiple different teraherz frequencies distributed between a minimum frequency and a maximum frequency, where the minimum and maximum frequencies are between about 0.5 THz and about 2 THz. Viruses can have a teraherz absorption band such that reflected electromagnetic waves are modified from incoming waves to form a unique signature response. Electromagnetic waves reflected from the sensor surface can be measured at the multiple different frequencies to form a measured reflection spectrum. The measured reflection spectrum can be compared to a spectrum indicating the presence of the target virus when the reflection spectrum is compared to the known unique signature response for a particular virus. The method can also include outputting a detection signal.

There has thus been outlined, rather broadly, the more important features of the invention so that the detailed description thereof that follows may be better understood, and so that the present contribution to the art may be better appreciated. Other features of the present invention will become clearer from the following detailed description of the invention, taken with the accompanying drawings and claims, or may be learned by the practice of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B schematically illustrate a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIGS. 2A and 2B schematically illustrate a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 3A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 3B is a top view of the colorimetric pathogen sensor of FIG. 3A.

FIG. 3C is an optical image detail view of a colorimetric pathogen sensor in accordance with an example of the sensor schematically illustrated in FIG. 3A when a pathogen is attached.

FIG. 4A is a schematic illustration of a colorimetric pathogen sensor before exposure to a virus in accordance with an example of the present disclosure.

FIG. 4B is a schematic illustration of the sensor of FIG. 4A after exposure to a virus in accordance with an example of the present disclosure.

FIG. 5A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 5B shows the colorimetric pathogen sensor of FIG. 5A when a pathogen is attached.

FIG. 5C is an optical image of a microfabricated optical grating colorimetric whole virus sensor.

FIG. 6 is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 7A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 7B shows the colorimetric pathogen sensor of FIG. 7A when a pathogen is attached.

FIG. 8A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 8B shows the colorimetric pathogen sensor of FIG. 8A when a pathogen is attached.

FIG. 9A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 9B shows the colorimetric pathogen sensor of FIG. 9A when a pathogen is attached.

FIG. 10A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 10B shows the colorimetric pathogen sensor of FIG. 10A when a pathogen is attached.

FIG. 11A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 11B shows the colorimetric pathogen sensor of FIG. 11A when a pathogen is attached.

FIG. 11C is an optical image of a colorimetric pathogen sensor in accordance with an example of the sensor schematically illustrated in FIG. 11A.

FIG. 11D is an optical image of a colorimetric pathogen sensor in accordance with an example of the sensor schematically illustrated in FIG. 11A.

FIG. 11E is an optical image of a colorimetric pathogen sensor in accordance with an example of the sensor schematically illustrated in FIG. 11A.

FIG. 11F is an optical image of the colorimetric pathogen sensor of FIG. 11E when a pathogen is attached.

FIG. 11G is an optical image of a colorimetric pathogen sensor in accordance with an example of the sensor schematically illustrated in FIG. 11A.

FIG. 11H is an optical image of the colorimetric pathogen sensor of FIG. 11G when a pathogen is attached.

FIG. 12A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 12B shows the colorimetric pathogen sensor of FIG. 12A when a pathogen is attached.

FIG. 13A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 13B shows the colorimetric pathogen sensor of FIG. 13A when a pathogen is attached.

FIG. 14A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 14B shows the colorimetric pathogen sensor of FIG. 14A when a pathogen is attached.

FIG. 14C is an optical image of a colorimetric pathogen sensor in accordance with an example of the sensor schematically illustrated in FIG. 14A when a pathogen is attached.

FIG. 15A-D schematically illustrates microbead amplification factors.

FIG. 16A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 16B shows the colorimetric pathogen sensor of FIG. 16A when a pathogen is attached.

FIG. 17A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 17B shows the colorimetric pathogen sensor of FIG. 17A when a pathogen is attached.

FIG. 18A is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 18B shows the colorimetric pathogen sensor of FIG. 18A when a pathogen is attached.

FIG. 19A illustrates the spectra of a blue spot on a peacock feather before and after adding a virus.

FIG. 19B shows a region of the peacock feather referenced in FIG. 19A that has the blue spot.

FIGS. 20A and 20B schematically illustrate a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIGS. 21A-21C show light polarization and light transmission changes of a colorimetric pathogen sensor in accordance with an example of the present disclosure

FIG. 22A is a graph of hydrogel transmission spectra shifted to smaller wavelengths as it shrank in PBS X1 buffer solution.

FIG. 22B is a difference spectrum clearly showing the shift of FIG. 22A.

FIGS. 23A and 23B schematically illustrate a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 24 is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 25 is a schematic illustration of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 26A is an optical image showing micro textured grating patterns surface fabricated using optical lithography and etching of platinum.

FIGS. 26B and 26C are optical images showing that the presence of viruses on a micro textured grating pattern changes the appearance of the grating lines and its effective color.

FIG. 27A is an SEM of silicon surface etched using DRIE and coated with gold.

FIG. 27B shows the surface of FIG. 27A additionally wet etched and coated with a dielectric layer.

FIG. 27C illustrates a pseudo-colored electric field intensity of a TE wave reflected at 28° projected onto a gold-coated silicon with 500 nm rms roughness.

FIG. 27D shows TE and TM mode reflections at 28° from as etched and dielectric-coated (aptamer and virus) surfaces.

FIGS. 28A-28C illustrate nanotextured surfaces produced by various techniques.

FIGS. 29A and 29B are schematic illustrations of a colorimetric pathogen sensor in accordance with an example of the present disclosure.

FIG. 30 is a flowchart illustrating another example method of detecting a virus in accordance with an example of the present disclosure.

FIG. 31 is a schematic of a teraherz frequency based sensor in accordance with an example of the present disclosure.

FIG. 32 is a schematic of another teraherz frequency based sensor in accordance with an example of the present disclosure.

FIG. 33 is a schematic of another example teraherz frequency based sensor in accordance with an example of the present disclosure.

FIG. 34 is a graph of reflection coefficient spectra for an example teraherz frequency based sensor in accordance with an example of the present disclosure.

FIG. 35 is a graph of reflection coefficient spectra for another example teraherz frequency based sensor in accordance with an example of the present disclosure.

FIG. 36 is a graph of reflection coefficient spectra for another example teraherz frequency based sensor in accordance with an example of the present disclosure.

These drawings are provided to illustrate various aspects of the invention and are not intended to be limiting of the scope in terms of dimensions, materials, configurations, arrangements or proportions unless otherwise limited by the claims.

DETAILED DESCRIPTION

While these exemplary embodiments are described in sufficient detail to enable those skilled in the art to practice the invention, it should be understood that other embodiments may be realized and that various changes to the invention may be made without departing from the spirit and scope of the present invention. Thus, the following more detailed description of the embodiments of the present invention is not intended to limit the scope of the invention, as claimed, but is presented for purposes of illustration only and not limitation to describe the features and characteristics of the present invention, to set forth the best mode of operation of the invention, and to sufficiently enable one skilled in the art to practice the invention. Accordingly, the scope of the present invention is to be defined solely by the appended claims.

Definitions

In describing and claiming the present invention, the following terminology will be used.

The singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a particle” includes reference to one or more of such materials and reference to “subjecting” refers to one or more such steps.

As used herein, the term “about” is used to provide flexibility and imprecision associated with a given term, metric or value. The degree of flexibility for a particular variable can be readily determined by one skilled in the art. However, unless otherwise enunciated, the term “about” generally connotes flexibility of less than 2%, and most often less than 1%, and in some cases less than 0.01%.

As used herein with respect to an identified property or circumstance, “substantially” refers to a degree of deviation that is sufficiently small so as to not measurably detract from the identified property or circumstance. The exact degree of deviation allowable may in some cases depend on the specific context.

As used herein, “adjacent” refers to the proximity of two structures or elements. Particularly, elements that are identified as being “adjacent” may be either abutting or connected. Such elements may also be near or close to each other without necessarily contacting each other. The exact degree of proximity may in some cases depend on the specific context.

As used herein, a plurality of items, structural elements, compositional elements, and/or materials may be presented in a common list for convenience. However, these lists should be construed as though each member of the list is individually identified as a separate and unique member. Thus, no individual member of such list should be construed as a de facto equivalent of any other member of the same list solely based on their presentation in a common group without indications to the contrary.

As used herein, the term “at least one of” is intended to be synonymous with “one or more of.” For example, “at least one of A, B and C” explicitly includes only A, only B, only C, and combinations of each.

Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limits of 1 to about 4.5, but also to include individual numerals such as 2, 3, 4, and sub-ranges such as 1 to 3, 2 to 4, etc. The same principle applies to ranges reciting only one numerical value, such as “less than about 4.5,” which should be interpreted to include all of the above-recited values and ranges. Further, such an interpretation should apply regardless of the breadth of the range or the characteristic being described.

Any steps recited in any method or process claims may be executed in any order and are not limited to the order presented in the claims. Means-plus-function or step-plus-function limitations will only be employed where for a specific claim limitation all of the following conditions are present in that limitation: a) “means for” or “step for” is expressly recited; and b) a corresponding function is expressly recited. The structure, material or acts that support the means-plus function are expressly recited in the description herein. Accordingly, the scope of the invention should be determined solely by the appended claims and their legal equivalents, rather than by the descriptions and examples given herein.

Colorimetric Pathogen Sensors

In general, as illustrated in FIG. 1A, a colorimetric pathogen sensor 100 as disclosed herein can comprise a substrate 110 and an molecular recognition group (e.g. aptamer) 120 coupled to the substrate 110. The molecular recognition group 120 can be operable to selectively bind to a target pathogen 130. Upon the molecular recognition group 120 binding with the target pathogen 130, as shown in FIG. 1B, reflected light can be altered thereby changing apparent color. The substrate 110 can comprise any suitable material, such as silicon material, a nitride material (e.g., silicon nitride), and/or a cellulose material. The substrate 110 can be opaque, translucent, or transparent, in whole or in part, with regards to light transmission. The substrate can also be patterned with the grating structure or other dispersive surfaces to enhance the visible changes that occur in the presence of the virus.

Different configurations of colorimetric sensors for detecting pathogens, such as whole viruses, bacteria, etc., can be visibly read using the naked eye without requiring any batteries or electronics, although color detectors can also be used. For example, a color detector may be oriented adjacent the substrate so as to register color changes.

Viral biomolecules can be any chemical entity which is directly or indirectly related to the presence of viruses by indicating a current or previous infection with the virus. Non-limiting examples of viral biomolecules can include whole virus, antibodies, antigens, viral proteins, viral RNA, viral DNA, viral biomarkers, and the like.

Molecular recognition groups can be any chemical compound or group that selectively binds with the target virus. Non-limiting examples of molecular recognition groups can include aptamers, antigens, antibodies, and the like.

Molecular recognition groups can generally include a surface bonding group and a virus binding group. Metal surfaces can be bonded with aptamers, antigens, antibodies, or other molecular recognition groups using any suitable functionalization technique. Further, metal surfaces can optionally be first prepared or activated via functionalization with an active group which binds with a corresponding end of the molecular recognition group. For example, a thiol group can be attached to the metal surface. However, in many cases, the molecular recognition group can include a surface bonding group which directly bonds to the metal surface. Non-limiting examples of surface bonding groups can include organosulfur thiols such as alkyl thiols, dialkyl disulfides, etc. Metal, e.g. gold, surface can also be functionalized via techniques such as, but not limited to, oligonucleotide functionalization via thiol groups, surface saturation with single stranded oligonucleotides, PEGylation optionally including thiol or azide bonding groups, photonic immobilization, azide functionalization, and the like. For some details on the known synthetic functionalization techniques, see INNOVACOAT Gold coatings; Polo E. et al. (2013) Tips for the Functionalization of Nanoparticles with Antibodies. In: Guisan J. (eds) Immobilization of Enzymes and Cells. Methods in Molecular Biology (Methods and Protocols), vol 1051. Humana Press, Totowa, N.J. pp. 149-163; Tiwari et al. Nanomaterials 2011, 1(1), 31-63, Functionalized Gold Nanoparticles and Their Biomedical Applications; which are each incorporated herein by reference. For example, thiols can be functionalized at one or both ends of an aptamer, an antigen, or an antibody.

In a specific example, the aptamers can include a thiol end group configured to bind with gold electrodes. Thiol end groups bind with almost any materials. Thiol end groups are very aggressive and in some cases may cause corrosion of some metallic surfaces. In those cases other functional end groups with lower binding energies can be used such as, but not limited to, metal-carbon (e.g. carbene, acetylide, vinylidene, etc), metal-nitrogen (e.g. nitrene, etc), azides, and the like.

Formation of a layer of molecular recognition groups can be performed using liquid phase deposition. A molecular recognition group (e.g. aptamer) solution can be applied to the surface in order to bond with the surface. The functionalized group can then react with the exposed surface to bind the molecular recognition groups to the surface. Residual unreacted materials can be removed by washing or evaporation.

The molecular recognition mechanism in the sensors can be based on aptamers that are oligonucleotides designed to bind to specific capsid proteins or nucleic acids on the surface of the target whole virus to be detected. The aptamers can be synthesized in-house using the known SELEX (systematic evolution of ligands by exponential enrichment) process or such aptamer preparation can be outsourced. In one specific example, the sensors can detect Zika virus. On another example, the aptamers can be selective for SARS-CoV-2. Regardless, with a suitable molecular recognition group, almost any other virus, bacteria and pathogen can be detected using these sensors. The surface proteins on most viruses and bacteria mimic the structure of sugars that are sought after by the living cells. Molecular recognition group selectivity can be reduced in targeting and binding to a very specific pathogen. To improve sensor selectivity, multiple molecular recognition groups can be used so that when most of them bind with a target, the mechanical forces generated on the sensor structure reaches the sensing threshold. Furthermore, multiple different molecular recognition groups which selectively bond with different functional groups of a virus can be used simultaneously on a common substrate or on adjacent sensors or substrates within an array of sensors. This approach can be used to increase sensor selectivity appreciably.

As a general guideline, some example sensor devices discussed herein take advantage of molecular recognition group and virus binding energies with substrates, microbeads, and with each other. Other example sensor devices rely on these binding energies as well as the refractive index of the molecular recognition group-virus complexes. Some example sensor devices may rely solely on the refractive index changes that are affected by the molecular recognition group-virus complexes. Examples of each of these mechanisms will be discussed in more detail below.

The colorimetric sensors disclosed herein can be used to detect nearly all the viruses, bacteria and pathogens if they can be functionalized with appropriate corresponding molecular recognition groups. It is noted that the following descriptions largely exemplify using aptamers and virus, although these same principles also apply to other molecular recognition groups and pathogens.

As an example, to immobilize and fix aptamers on sensor surfaces, aptamers with thiol end group can be used that readily attach to gold and other metals, most polymers and plastics. For example, the thiol binding with cellulose acetate is as strong as thiol-gold binding of ˜1-2 eV and results in a rupture force of ˜0.5-1 nN. In the specific case of Zika virus, the aptamer-Zika binding energy depends on the Zika surface condition (deactivated Zika tested had an uneven surface) and can have binding energies ranging from 0.05 eV to 1 eV. The binding energies between the aptamer, the substrate, and the pathogen (e.g., Zika or other virus, bacteria, etc.) play an important role.

In one aspect, the aptamer 120 can be coupled directly (e.g., bonded via covalent, ionic, or other attractive forces) to the substrate 110 or the aptamer 120 can also be coupled to the substrate 110 via an intermediate structure. For example, as illustrated in FIG. 2A, a colorimetric pathogen sensor 200 can include a substrate 210, an aptamer 220, and a base 240. The aptamer 220 can be coupled to the substrate 210 via the base 240. In particular, the base 240 can be bonded to the substrate 210 by a first bond 250, and the aptamer 220 can be bonded to the base 240 by a second bond 251. The aptamer 220 can be operable to bind to a target pathogen 230 by a third bond 252, as shown in FIG. 2B. The configuration and various aspects of the colorimetric pathogen sensor 200 are illustrated generally with regard to FIGS. 3A-8B.

The base 240 can comprise any suitable material, such as a metal material (e.g., gold, aluminum, and/or nickel), a polymer material (e.g., cellulose acetate), a ceramic material, composites of these materials, or alloys thereof, in any suitable configuration. In one aspect, the base 240 can comprise a coating or layer substantially covering a surface of the substrate. For example, the substrate working surface can be coated with a base layer, such as a layer of gold or other suitable metals or a layer of polymers. The aptamers can functionalize the base or coated surface with a large binding energy. The coating material plays an important role in immobilizing the aptamers. In one specific example, a thiol end group can be used to attach aptamers to gold, cellulose acetate, and other polymers. Other functional end groups can be used to bind directly to a working surface, such as in a cantilevered beam or other structure.

In one aspect, the second bond 251 may generate a residual stress on the base 240 insufficient to separate the base 240 from the substrate 210. In another aspect, the substrate 210 can be configured to bend under residual stress generated by attachment of the pathogen via the third bond 252. The substrate 210 can have any suitable configuration to achieve such bending, such as a beam configuration (e.g., a cantilever beam or a flexure as illustrated in FIG. 3A). Thus, upon the aptamer 220 binding with the target pathogen 230, the third bond 252 can generate or transfer a residual stress on the base 240 that causes the substrate 210 to bend (e.g., in direction 260 in FIG. 2B), which can alter reflected light and thereby change apparent color. The substrate 210 configured as a beam can therefore deflect when the base 240 and attached surface aptamers 220 bind to the pathogen 230, which can alter reflected light and change color to indicate the presence of the pathogen 230 to an observer or external sensor (e.g., an optical sensor). The base or coating material can be selected to adjust the residual stress on the sensor surface. The residual stress of the surface layer and the aptamer on the surface can bend the cantilever beam from its original shape. The binding force to the pathogen can range from a tenth of an electron volt to few electron volts and can bend the cantilever beam by a larger amount. The cantilever beam can be designed so that its bending can be seen directly by the naked eye and can be measured using a graded scale behind the beam as illustrated in FIGS. 3B and 3C. In one aspect, the cantilever beam can be equipped with an optical indicator (e.g., a colored patch on its tip). For example, as shown in FIG. 4A-4B, when the beam bends, a colored tip can move and reveal its location against a suitable background color.

In another display mechanism, the base 240 may be configured to provide optical or refractory changes when the substrate 210 bends. For example, as illustrated in FIGS. 5A and 5B, the base can comprise a grating (e.g., an optical grating) that forms parallel strips on a surface of the substrate (e.g., the cantilevered beam). Thus, a cantilever beam can be equipped with a grating on its surface that reflects the incident light differently when a pathogen is present. FIG. 5A shows a schematic of optical gratings on multiple cantilever beams, which bend upward when a pathogen is attached, as shown in FIG. 5B. FIG. 5C shows photograph of a microfabricated cantilever beam with patterned gold layers (e.g., a refraction grating) reflecting light differently when its surface aptamers bind to a pathogen. Typical cantilever materials are silicon nitride, silicon, silicon dioxide, polysilicon, polyurethane, polyvinylidene difloride (piezoelectric), and many other materials and polymers. The devices shown in FIG. 5C were fabricated using 100 nm thick silicon nitride cantilever beams and the beam ranged in length from 100 um up to 600 um and they were 50 um wide.

In one aspect, the base may not cover an entire working or functional surface of the substrate and therefore some surfaces of the substrate may be exposed. For example, the base may not be applied to, or cover, the opposite side of the substrate (e.g., the base may only be applied to one side of the substrate) as in FIG. 3A, and/or the substrate may be exposed between gratings as in FIG. 5A. In such cases, the aptamer may bond with the exposed surface portion of the substrate not covered by the base.

In another example, the substrate (e.g., a cantilever beam) can be configured to bend so as to delaminate from an ink well substrate and release a visible dye or ink from an ink well, as shown in FIG. 6 . Alternatively, ink wells or reservoirs can be covered with a base (e.g., a gold protective layer) with an attached aptamer. The ink well can release ink when a pathogen attaches to the aptamer on the base (e.g., gold layer), which can delaminate the base from the substrate. This alternative aspect is discussed in more detail below with regards to FIGS. 11A-13B. Adhesion between the substrate and the base can be adjusted by thickness of the adhesion layer (chromium, nickel, indium, etc.). For example, 1 nm to 40 nm chromium as adhesion layer on silicon, silicon dioxide and silicon nitride can be used. At 1 nm, the adhesion forces are very small and the gold/chromium layer can be easily peeled off using a scotch tape. Many parameters including the residual layers on the starting surface and the deposition method (e.g. evaporation, sputtering, atomic-layer deposition, or the like) and conditions (e.g. temperature, vacuum level, etc.) all affect and contribute to the adhesion forces. Sputtered chromium around 10 nm thickness was used in the examples above.

In some examples, a cantilever beam can be configured with two different colors on the front and back surfaces, as illustrated in FIG. 7A. When the beam bends as a pathogen is attached at the surface, the apparent color can change and become completely different, as schematically shown in FIG. 7B. This color change can be displayed by virtue of beams curling while viewing angles for color display can also be adjusted to improve visualization of the color response.

In another alternative mechanism, cantilever beams can be operable to block passage of light prior to the aptamer binding with the target pathogen, as illustrated in FIG. 8A. These devices can also transmit light upon the aptamer binding with the target pathogen and bending of the substrate (e.g., when viruses are attached onto their surfaces), as illustrated in FIG. 8B. FIGS. 8A and 8B illustrate a schematic of a “light valve” colorimetric sensor. Absorption of a pathogen results in the deformation of the cantilever beams and the amount light that passes through the sensor increases considerably by opening a gap between adjacent rows of cantilevered beams. Such differences in light intensity can be detected visually or by optical sensors oriented to measure the amount of light passing through the light valve opening.

In some examples, as illustrated in FIG. 9A, the substrate (e.g., beam or membrane) can be supported at opposite ends to form a bridge. As illustrated in these figures, the beam or membrane can comprise a transparent material (e.g., silicon nitride) and the base can comprise a dispersive element on a surface of the beam. In the illustrated example, the dispersive element can comprise a grating forming parallel strips on a surface of the beam or membrane. In addition, the sensor can include a reflective bottom surface or membrane (e.g., bottom reflective gold layer) disposed below the beam or membrane bridge. As one example, this configuration can form a Fabry-Perot resonator in which a variable gap distance between the bridge and bottom surface indicates presence of a virus. As illustrated in FIG. 9B, when a pathogen is present and binds to the surface aptamers, the beam or membrane can deflect and deform, changing the reflected color or completely eliminating the color. In one example, the distance between a semi-transparent beam and the reflecting bottom surface can be used to create a reflection-mode Fabry-Perot resonator that only reflects blue. When a pathogen is present, it can change the refractive index inside the resonator and change the reflected color to yellow or red, for example. The specific refractive index, degree of bridge deflection, and reflective bottom surface can all be varied to achieve different color responses.

Many variations of the resonator approach can be realized using periodic reflecting surfaces and nanocrystals (e.g., photonic crystals) with different absorption spectra, etc. One example is the chameleon skin color changing mechanism as a biomimetic device adopted to sense pathogens as schematically shown in FIGS. 10A and 10B. In this case, the chameleon mimetic colorimetric sensor is configured as a reflection-mode Fabry-Perot resonator that can include dispersive surfaces permitting two different colors (e.g., wavelengths) to be transmitted/reflected through the Fabry-Perot resonator, which is tuned to only reflect one of the wavelengths (e.g., blue) when a pathogen is not present (FIG. 10A). For example, the sensor can include a first dispersive surface (e.g., red photonic crystals) on a surface of the beam or membrane bridge and a second dispersive surface (e.g., blue photonic crystals) on the reflective bottom surface or membrane. The dispersive surfaces can be operable to reflect different wavelengths of light (e.g., red or blue). When a pathogen is present and binds to the sensor's surface aptamers, the binding force creates a surface stress causing the Fabry-Perot length (L) to increase and reflect the red light (FIG. 10A). The dispersive surfaces such as photonic crystals can be formed by additive manufacturing, photolithography, direct laser writing/drilling, self-assembly, e-beam lithography, nano-imprinting, and the like. One very inexpensive technique would involve using aptamers to assemble patterned nanoparticles to form the photonic crystal. In this case, two different types of aptamers will be used: one for binding with the virus, and another one to DNA-template the formation of the photonic crystal.

Referring again to FIGS. 2A and 2B, in one aspect, the second and third bonds 251, 252 can be stronger than the first bond 250. In this case, upon the aptamer 220 binding with the target pathogen 230, the third bond 252 can generate a residual stress on the base 240 that causes the base 240 to separate from the substrate 210 thereby altering reflected light and changing apparent color. In some examples, this type of sensor may rely on patterned substrates (e.g., a base pattern on a substrate) where the pattern changes or disappears in the presence of the pathogen. The configuration and various aspects of this type of colorimetric pathogen sensor are illustrated generally with regard to FIGS. 11A-13B.

The base 240 can have any suitable configuration, such as a plurality of pads or detachable “islands” arranged on a surface of the substrate (see, e.g., the gold dots of FIGS. 11A and 11E). The plurality of pads can have any suitable configuration, such as at least one of a circular configuration (FIG. 11A), a triangular configuration (FIG. 11C), or a rectangular configuration (FIG. 11D). In a particular example, the base can comprise gold dots with carefully selected dimensions (e.g., thickness, width, and/or diameter) on silicon dioxide or any other substrate with small adhesion forces to gold. For example, silicon dioxide has very small adhesion force with gold (e.g. Hamaker coefficient is small resulting in small van der Waals/Casimir forces). Including a thin (1 nm-40 nm) metallic layer such as chromium between the gold and the silicon dioxide layer, the adhesion force can be modified and changed from a very small value to relatively large value when chromium thickness is around 40 nm. Aptamers can then be added to the gold surface. Aptamer-gold binding generates residual stresses that are not sufficiently large enough to completely peel-off the gold dot from the substrate. However, in the presence of the pathogen and the residual stress provided by the aptamer-pathogen bond, the residual stress exceeds the value required to peel-off the gold dot and leaves behind the bare substrate. Once the gold dots peel off (see, e.g., FIGS. 11B and 11F), the apparent color completely changes. As shown in FIG. 11F, fewer than all of the plurality of pads (e.g., gold dots) may be caused to separate from the substrate. In this case, the color change is easily detected visually by the loss of a significant portion of the gold islands. FIG. 11G illustrates the above-described concepts utilizing gold beams (horizontal) connected along chromium bars (vertical). After Zika virus bonded with the aptamer, the beams are peeled off, as illustrated in FIG. 11H. This same approach can be used with other pathogens by selecting the appropriate aptamer.

In one aspect, the base 240 can comprise a plurality of microbeads (see, e.g., FIG. 12A), which can be arranged on a surface of the substrate. In this case, the microbeads can be immobilized (e.g., mildly glued) on the sensor surface. For example, suitable aptamers can be used to bind the microbeads to the surface. Alternatively, one can use surface forces to mildly attach the beads to the surface. In another alternative, electrostatic forces that are readily available at that scale can be used. When a pathogen is introduced, the pathogen can bind with aptamers on the microbeads. The resulting pressure can separate or release the microbeads from the substrate (see, e.g., FIG. 12B). The released microbeads result in a change in color of the substrate and/or the fluid above the substrate. For example, microbeads may be a metal, polymer, or ceramic material which exhibit a particular color. Upon removal the underlying substrate is no longer layered with the microbeads such that the native substrate color would then be visible.

In some examples, a sensor can include an ink well formed in the substrate and ink can be disposed in the ink well. The ink can be covered by the base (e.g., pads or the gold dots of FIG. 13A) and upon separation of the base from the substrate the ink can be released from the ink well (FIG. 13B). In a particular example, reservoirs of liquid or powder ink under gold dots or other protective layers composed of appropriate metals (e.g., aluminum, nickel, etc.) can be sandwiched between thin layers of polymers (e.g., polystyrene). When the surface residual stress generated by aptamer/virus attachments become sufficiently large, the gold dots or other protective layers can peel-off and release the ink, completely changing the color of the fluid on the device surface. As in one example, when a pathogen binds to an aptamer-functionalized gold protective layer over the ink well, this bond can generate large residual stress to overcome the gold-substrate stiction force. The ink or dye under the gold protective layer can then be released, changing the color of a liquid in proximity to the sensor or included with the sensor.

Referring again to the colorimetric pathogen sensor 100 of FIG. 1A, in some examples, the aptamer 120 can be bonded (e.g., directly) to the substrate 110. In one aspect, such a sensor can include a microbead (e.g., a colloidal bead) bonded to an aptamer, as shown in FIG. 14A. In this case, the aptamer on the substrate and the aptamer on the microbead can bind to the target pathogen, as shown in FIG. 14B. Thus, in the presence of the virus, the microbeads can aggregate and stick to the sensor surface and change its color, as illustrated in the photograph of FIG. 14C. The microbeads can be colored (e.g., bright red) to enhance visibility. Microbeads usually come from commercial suppliers with surfactants to prevent them from agglomerating with each other.

Microbeads can provide an amplification mechanism rarely seen in other tags. FIG. 15A-D shows the microbead interaction mechanisms and its amplification factor. For 100% surface coverage by aptamers, viruses and color tags, the microbead amplification factor will be around 1×. However, if the surface coverage by aptamers is small and even smaller fractions of these aptamers are tagged by viruses and microbeads, then larger beads provide large amplification factor proportional to their radius as schematically shown in FIGS. 15B and 15D. Forces acting on larger beads, however, inside liquids can be problematic. Shear forces on microbeads in liquids arising from convective and other currents is proportional to their surface areas or ˜R² while buoyancy is proportional to their volume or ˜R³. The stiction force is proportional to their surface area in contact with the substrate that also changes as R². FIG. 15C shows that there is a cross-over point between these forces as a function of the microbead radius that indicates the most optimum bead radius for specific binding energy, surface coverage and bead material density. For visible light viewing and detection using the naked eye, it may be desirable to use microbeads larger than 0.5 μm in diameter. In some examples, a diameter of the microbead can be from about 0.5 μm to about 10 μm. In particular examples, a diameter of the microbead can be from about 1 μm to about 4 μm.

In some examples, colorimetric pathogen sensors can include a plurality of bases (e.g., microbeads), and an aptamer bonded to the plurality of bases, as shown in FIG. 16A. Upon the aptamer binding with the target pathogen, the plurality of bases can group together via the bonds to the aptamer, as shown in FIG. 16B, thereby altering reflected light and changing apparent color. In one aspect, the bases can comprise microbeads that form a colloidal solution and coagulate when the pathogen is introduced. The pathogen can act as a bridge attaching to the aptamers of many beads and bringing them together. In this example, the microbeads can be used in any container and a fixed sensor structure is not needed since the agglomeration of microbeads will form a visible mass.

In another example, the substrate can comprise a plurality of beams (e.g., a biomimetic compliant metamaterial forming scales) functionalized with aptamer, as shown in FIG. 17A. The beams or scales can be caused to bend by a binding force of the aptamer and the target pathogen moving the target pathogen into a gap between at least two of the plurality of beams or scales, as schematically shown in FIG. 17B. In other words, when the pathogen is present, the pathogen-aptamer binding force can push the pathogen into the structure increasing the gaps between beams or scales. Such separation between two beams or scales can modify the optical patterns presented by the beams or scales and can be used to create colorful reflections under white light as elegantly displayed by the peacock feather. When the pathogen is absorbed by the aptamers on the beam or scale surfaces, the generated residual stress changes the separation of the surfaces and the color patterns of the reflected wave. As a non-limiting example, these photonic crystal metamaterials can be fabricated using electron beam lithography and top-down etching techniques. Other techniques can include bottom up techniques or a combination of top-down and bottom-up techniques.

In one aspect, the effective refractive index surrounding the structures can also be changed. Sensor function can be based on the change in refractive index of the aptamer surface layer when the pathogen is targeted. In this case, the change in the reflected colors can be caused by the change in the refractive index of the surface-absorbed layers as shown in FIGS. 18A and 18B which are micrographs. FIG. 18A shows a biomimetic material (e.g., a substrate comprising at least one of microstructures or nanostructures) with blue appearance under white light. When a pathogen binds to the surface aptamer coating, the appearance and color changes, as shown in FIG. 18B having a red appearance.

FIGS. 19A and 19B show the effect of aptamer/virus (Zika in this example) on the reflected light from a peacock feather with microstructures shown in FIGS. 17A and 17B. The aptamer/virus completely changes the reflected peak wavelength and local color. In FIG. 19A, spectra of a blue spot on the peacock feather before and after adding the virus (in this case Zika) on a region with aptamer shown inside the circle in FIG. 19B. The Zika/aptamer shifts the reflected spectrum to higher wavelengths indicating that addition of Zika has increased the distance between the scales or flaps in FIG. 17B and/or it has increased the refractive index from n-1 corresponding to dry air to n˜1.17 corresponding to water with aptamer and Zika (in this case). Zika alone changes the refractive index by Δn˜0.01.

In some examples, as illustrated in FIG. 20A, a colorimetric pathogen sensor 300 can comprise a matrix 370 and an aptamer 320 embedded in the matrix 370, and a plurality of particles 380 embedded in the matrix 370. The aptamer 320 can be operable to bind to a target pathogen 330. Upon the aptamer 320 binding with the target pathogen 330, as shown in FIG. 20B, the matrix 370 can expand and change the position of the plurality of particles 380 relative to one another, thereby altering reflected light and changing apparent color.

In one aspect, the plurality of particles 380 can comprise nanoparticles, which can be made of any suitable material, such as gold. Nano-metallic, nano-dielectric and nano-semiconducting materials have electromagnetic properties that sensitively depends on their separation distances. Functionalization with aptamers and the subsequent absorption of viruses modulates the distance between the nanoparticles and changes the dielectric properties of their intervening regions as schematically shown in FIGS. 21A-21C. For example, as shown in FIG. 21A, depending on the orientation of the light polarization with respect to the NP-NP axis, the plasmonic interaction can result in red-shift (E∥R) or blue shift (E⊥R). As shown in FIG. 21B, gel expansion of 25% (ΔL/L˜0.25) should easily result in 25% increase in transmitted light intensity. As shown in FIG. 21C, nanoparticles can be deposited more regularly by setting up ultrasonic standing waves inside the gel before it sets. The pressure nulls will be separated by λ_(acoustic)/2˜35 μm in most gels at 100 MHz.

The matrix 370 can comprise any suitable material, such as a gel material (e.g., a hydrogel material), a paper material (e.g., cellulose), or any other suitable matrix material in which the aptamer can be embedded. For example, the aptamer can be embedded in cellulose (e.g., paper) or gels and hydrogels to enable trapping and binding of viruses. In a particular example, the colorimetric pathogen sensor 300 includes an aptamer-based hydrogel with embedded particles. In these scaffolding and structural matrix materials, the binding of the aptamer with a specific virus or pathogen may result in a dimensional change that can in turn can be used to implement a color change. FIGS. 22A and 22B show an example of the effect of structural change on the color of a hydrogel with embedded gold nanoparticles. FIG. 22A is a graph of hydrogel's transmission spectra shifted to smaller wavelengths as it shrank in PBS X1 buffer solution. FIG. 22B shows a difference spectrum clearly showing the shift. Optical images of the hydrogel in water and in PBS X1 are shown inset in FIG. 22A.

In some examples, as illustrated in FIG. 23A, a colorimetric pathogen sensor 400 can comprise first and second transparent plates 411, 412, a matrix 470 associated with the first and second plates 411, 412, and an aptamer 420 embedded in the matrix 470. The aptamer 420 can be operable to bind to a target pathogen 430. Each transparent plate can have a dispersive surface. Upon the aptamer binding with the target pathogen, as shown in FIG. 23B, a refractive index of the matrix can change, thereby altering reflected light from the dispersive surfaces and changing apparent color. In one aspect, the first and/or second plates 411, 412 can be similar to a substrate described herein.

In one aspect, the matrix 470 can be disposed between the first and second transparent plates 411, 412. Upon the aptamer 420 binding with the target pathogen 430, the matrix 470 can expand and change a distance 461 between the first and second transparent plates 411, 412, thereby altering reflected light from the dispersive surfaces and changing apparent color. The combination of distance and refractive index changes caused by pathogen binding to aptamers can be used to realize colorimetric virus and pathogen sensors.

FIG. 24 shows schematic of a distance modulation colorimetric sensor where pathogen binding results in the modulation of distance between two dispersive surfaces that change the color of the reflected light. For example, pathogens binding to the aptamers from both sides can cause the distance between two plates to decrease and change the transmitted/reflected color. The dispersive surface can comprise any suitable configuration or structure, such as a grating forming parallel strips. The first and second transparent plates can comprise any suitable material, such as silicon nitride, silicon dioxide, and a variety of polymers, and the like.

In one aspect, the first and second transparent plates 411, 412 can be disposed in the matrix 470 and maintained at a fixed distance 461 from one another. For example, FIG. 25 schematically shows a colorimetric sensor that changes color when the refractive index in its sensing region is modified. In particular, pathogen binding can result in the modulation of the refractive index between two dispersive surfaces that change the color of the reflected light. This sensor is very similar to the Fabry-Perot refractive index to color converter discussed above, but has additional dispersive wavelength selective regions to emphasize a particular color. The same principle is observed in a chameleon skin.

As discussed above with regard to the colorimetric pathogen sensor 100 of FIG. 1A, the aptamer 120 can be bonded (e.g., directly) to the substrate 110. In one aspect, the substrate 110 can comprise at least one of microstructures or nanostructures. The at least one of microstructures or nanostructures can comprise at least one of microtextured surfaces or nanotextured surfaces. Nanotextured and microtextured surfaces have unique appearance that can be designed to completely change when viruses are attached. FIGS. 26A-26C show a microfabricated grating. In particular, FIG. 26A illustrates a microtextured (e.g., grating pattern) surface fabricated using optical lithography and etching of platinum. As shown in FIGS. 26B and 26C, the presence of a pathogen (in this case Zika) changes the appearance of the grating lines and its effective color. FIG. 26C also shows the presence of crystalline features that grow around a spot with Zika. Such debris can be removed by washing the device after a few minutes of exposing it to a Zika solution.

FIGS. 27A-27D show a nanotextured surface with random features and a microtextured surface with random features as well. In particular, FIG. 27A shows a SEM of silicon surface etched using DRIE and coated with gold. FIG. 27B shows the same surface additionally wet etched and coated with a dielectric layer (e.g. silicon dioxide). FIG. 27C illustrates a pseudo-colored electric field intensity of a TE wave reflected at 28° projected onto a gold-coated silicon with 500 nm rms roughness. FIG. 27D illustrates TE and TM mode reflections at 28° from as etched and dielectric-coated (aptamer and virus) surfaces. Dielectric coating eliminates the large variations in the electric field. Such coating can also modify the wavelength selective surface and modify the reflected color of the light. In one aspect, the addition of aptamer and its corresponding pathogen can change nanotextured and microtextured surface appearances by providing a dielectric coating. Finite element simulations show the effect of a dielectric coating in reducing field non-uniformity that results in an apparent smooth surface.

Nanotextured surfaces can be produced by many different techniques. Nanotexturing can be accomplished using roughening etches (deep-reactive ion etching, electro-chemical oxidation/reduction etching, etc.) shown above, or using diffusion-limited aggregation and structuring shown in FIGS. 28A-28C, specifically, surface diffusion of gold on tungsten (FIG. 28A) and surface roughening by electrochemical etching (FIG. 28A). FIG. 28C illustrates surface diffusion of gold on tungsten after very long (5 hours) gold surface diffusion time at 700 C. The star-like gold clusters are around 100 μm in diameter. Sharp edges and features produced by surface roughening, nanotexturing, etc., can act as electric field enhancing antenna and increase light-matter interaction by a factor proportional to |E|⁴. This mechanism can also be used to change the reflected light's color from these surfaces in the presence of certain chemicals.

It should be recognized that any colorimetric pathogen sensor as disclosed herein can be modified with any appropriate aptamer to sense corresponding virus, pathogen, associated protein, and/or bacteria.

As an example of using the colorimetric sensors herein, fluid samples containing a suspected virus or pathogen can be directly placed on the sensor. These fluid samples can be diluted bodily fluids or undiluted samples (e.g. mucus, sputum, urine, etc). In one aspect, a colorimetric pathogen sensor as disclosed herein can work well in detecting viruses in their natural environment (e.g., in blood, urine, or other bodily fluid). Consistent sensor output regardless of the bodily fluid can be obtained by including filters and/or pH-balancing salts in the structure of the sensor system to accommodate fluid environments with different pH levels (e.g., acidity or alkalinity).

In one aspect, a colorimetric pathogen sensor as disclosed herein can be disposed in an enclosure that is field deployable. For example, FIGS. 29A and 29B show a simple structure with the filters and a wick used in one of its shallow wells and the microbeads coated with aptamers in the second shallow well. When the pathogen inside bodily fluid is introduced into the input well with the filters, its large particles are filtered and its pH is adjusted before osmotically diffusing into the second output well. In the output well, the colloidal microbeads with appropriate aptamers bind to the cover window that is also coated with aptamers in the presence of the pathogen making the window the color of the microbeads, which can enable detection of the pathogen with the naked-eye and without the need for additional sensors or instrumentation.

Another variation of colorimetric based biosensors can detect a target virus through reflected electromagnetic waves. In some examples, the electromagnetic waves used to detect the target virus can be in the terahertz (THz) range. The electromagnetic waves can have a frequency in the range of about 0.5 THz to about 2 THz in certain examples. Electromagnetic waves in this range can provide a good balance between penetration depth and spatial resolution for biological applications.

Many viruses can exhibit different reflection spectra when electromagnetic energy in this range is applied. For example, a reflection spectrum can be measured by directing electromagnetic energy toward a surface having viruses and aptamers bound thereon and measuring the amount of reflected energy. More specifically, a series of different frequencies can be used. Electromagnetic waves at the different frequencies can be directed at the surface having viruses and aptamers bound thereon and the reflection coefficient can be measured at the different frequencies. A reflection spectrum can be formed from the data. The reflection spectrum can show the reflection coefficient at the multiple different frequencies distributed across a certain frequency range. In some examples, the reflection spectrum of a surface without any viruses bound thereon can be different from the reflection spectrum of the surface with viruses bound thereon. Therefore, a target virus can be detected by comparing the reflection spectrum of the surface when an unknown sample is applied to the surface and the spectrum of the surface when the target virus is known to be present on the surface. If the reflection spectrum matches the known spectrum of the surface when the target virus is present, then the test result can show that the target virus is present. If the reflection spectrum matches the known spectrum of a clean surface, without any target virus, then the test result can show that the target virus is not present.

With this description in mind, FIG. 30 is a flowchart illustrating an example method 500 of detecting a virus. The method includes: exposing 510 a sensor surface to a fluid sample containing a suspected virus; irradiating 520 the sensor surface with electromagnetic waves at multiple different frequencies distributed between a minimum frequency and a maximum frequency, where the minimum and maximum frequencies are between about 0.5 THz and about 2 THz; measuring 530 reflected electromagnetic waves from the sensor surface at the multiple different frequencies to form a measured reflection spectrum; comparing 540 the measured reflection spectrum to a spectrum indicating the presence of the target virus; and outputting 550 a detection signal.

In various examples, the sensor surface can be modified with aptamers in the same ways as described above. The surface on which the aptamers are immobilized can include a variety of materials. In certain examples, the surface can be polystyrene, polycarbonate, graphene, metal, or other materials. In one example, the surface can include a gold coating for attaching the aptamers through sulfur linking groups.

FIG. 31 shows a particular example aptasensor 560 that includes a sensor surface 565 that is a surface of a polystyrene substrate 570. A sample fluid 575 that includes a suspected virus and aptamers that selectively bind to the virus is placed on the sensor surface. In this example, spacers 580 are placed on the sensor surface and a cover 585 is placed over the spacers and the sample fluid. A teraherz horn antenna 590 is positioned to direction a beam of teraherz waves in the range of 0.5 THz to 2 THz at the sensor surface. the terahertz frequency is selected to coincide with certain resonances and absorption band of the virus. An optional frequency multiplier can be connected to the horn antenna in order to increase the output frequency. For example, a frequency multiplier or extender can be connected to a rectangular waveguide as a teraherz frequency source. This allows for an input frequency of 8-40 GHz, for example, to be ramped up to the target teraherz frequency. The same teraherz frequency source can also simultaneously function as an antenna, or a separate dedicated teraherz antenna can be used. The horn antenna can also receive reflected waves. The antenna can be connected to a network analyzer to measure a reflection spectrum over a span of frequencies.

The spacers and cover can be used to contain the sample fluid in a specific volume under the cover. This can ensure a uniform thickness of the sample fluid on the substrate. In some examples, the absorption of the teraherz waves by the virus and aptamer can be due to the permittivity function of the virus and aptamer, and not due to resonant absorptions. Therefore, the absorption can depend on the amount of virus and aptamer in the path of the teraherz beam, and this can depend on the thickness of the sample fluid on the sensor surface.

Therefore, the spacers and cover can be used to obtain a uniform and known thickness of the sample fluid. The thickness of the spacers can be selected depending on the desired thickness of the sample fluid. In some examples, the spacers can have a thickness from about 10 micrometers to about 50 um. Spacers can be made from any terahertz transparent suitable material, such as Teflon, rubber, plastic, thin glass, and others. In further examples, the cover can be made from plastic, glass, or another material. In certain examples, the sensor surface can be modified with the aptamers before the sample fluid is placed on the sensor surface. In other examples, the sample fluid can include viruses and aptamers mixed together, and this sample fluid can be placed on the sensor surface. In this case, the aptamers can be freely dispersed within the sample fluid, or bonded to a dispersed particle, e.g. nanoparticles or microbeads. Most viruses have characteristic vibrations at terahertz range of frequencies. Thus, terahertz can be used to selectively detect viruses with molecular recognition of their vibrational modes.

FIG. 32 shows another example aptasensor 600. This example includes a teraherz horn antenna 660 as in the previous example. The substrate used in this example is a graphene layer 612. One surface of the graphene layer is used as the sensor surface 610. A sample fluid 630 is placed on the sensor surface where the sample fluid can be irradiated by the teraherz antenna. In some examples, the graphene layer can be modified with aptamers to bind the virus. In other examples, viruses can be detected in the sample fluid without using aptamers.

FIG. 33 shows a more complex aptasensor 700 that includes a sensor surface 710 and microbeads 770 that have been modified with aptamers 720. In this example, the sensor surface includes a gold layer 712 on a polycarbonate substrate 714. The gold layer is modified with aptamers. A sample fluid is placed on the sensor surface, and at the same time the microbeads modified with aptamers are mixed with the sample fluid. The aptamers on the sensor surface can bind to viruses 780 and the aptamers on the microbeads can also bind to the viruses at the same time. Thus, the viruses can act as a bridge between the sensor surface and the microbeads. Excess microbeads and aptamers can be removed by rinsing with deionized water. As in the previous examples, a teraherz horn antenna can be directed at the sensor surface. The teraherz horn antenna can be used to irradiate the sensor surface with teraherz electromagnetic waves and to receive reflected waves. As previously described, the antenna can be connected to a network analyzer that can be used to record a reflection spectrum of the sensor surface.

In some examples, the sensor surface can be a flat surface. In other examples, the sensor surface can include wells or depressions to contain sample fluids. Wells can be any shape, such as square, rectangular, hexagonal, circular, and so on. As a general guideline, the width of the wells can vary from about 0.1 mm to about 10 mm.

In these teraherz example aptasensors, a target virus can be detected based on the reflection spectrum measured using the teraherz antenna. In some examples, the reflection coefficient (which is S-parameter S11) can be measured at multiple frequencies across a span of frequencies. The reflection coefficient measurements at these frequencies can make up a spectrum. In certain examples, the target virus can have a characteristic frequency at which the minimum reflection coefficient occurs. For example, the Zika virus can have a minimum reflection coefficient at 1.073 THz and a Zika virus bound to an aptamer can have a minimum reflection coefficient at 1.064 THz. Therefore, the Zika virus or the Zika/aptamer combination can be detected by measuring a reflection spectrum that has a minimum reflection coefficient at one of these frequencies. In further examples, sample fluids with known amounts of a particular target virus can be tested using the aptasensor and the reflection spectrum can be recorded. When unknown sample fluids are tested, the reflection spectrum of the unknown sample fluid can be compared to the previously record spectrum.

The span of frequencies over which the reflection coefficient is measured can be any span in the range of about 0.5 THz to about 20 THz, and in some cases 0.5 THz to 2 THz. In some examples, the span can include frequencies from a minimum frequency to a maximum frequency. The difference between the minimum and maximum frequencies can be from about 0.01 THz to about 1 THz, or from about 0.05 THz to about 0.5 THz, in some examples. The span that is measured can be selected to encompass the characteristic frequency of the minimum reflection coefficient for the target virus. The span can also encompass the frequency of the minimum reflection coefficient for the sensor surface when no virus is present. In a specific example, a sensor for Zika virus can use a span from about 1.0 THz to about 1.1 THz.

The reflection coefficient can be measured at a number of different frequencies between the minimum frequency and maximum frequency. The number of frequencies measured can be sufficient to provide a desired level of resolution in the reflection spectrum. In some examples, the frequencies can be from about 1/1000^(th) of 1 THz apart to about 1/10^(th) of 1 THz apart, or from about 1/1000^(th) of 1 THz to about 1/100^(th) of 1 THz apart. The reflection coefficient depends on the material permittivity/permeability and the thickness of the viral layer. By using spacers, we fix the viral layer thickness. The amount of target virus present can also be correlated with the minimum reflection coefficient (e.g. via magnitude and/or location).

The sensitivity of the aptasensor can be expressed in terms of Hz per individual virus. In some examples, the aptasensors that utilize teraherz electromagnetic waves can have a sensitivity from about 50 Hz/virus to about 200 Hz/virus or from about 50 Hz/virus to about 100 Hz/virus.

Example 2 Detecting Zika Virus through Teraherz Wave Reflection

Aptamers that bind specifically to the SF9 envelope protein of the Zika virus were obtained from BasePair Biotechnologies, Inc. The aptamers included thiol groups for bonding to the sensor surface. The aptamers were activated by resuspending the aptamers in a resuspension buffer provided by BasePair Biotechnologies, Inc. The aptamer solution was then diluted to 100 micromolar working concentration using Aptamer Folding Buffer from BasePair Biotechnologies, Inc., and the solution was heated to a temperature from 90° C. to 95° C. for 5 minutes and cooled to room temperature for 15 minutes. The 100 micromolar solution was then further diluted to 1 micromolar using buffer solution. The buffer solution was prepared with 1× concentration of Phosphate Buffer Saline and 1 a millimolar concentration of magnesium chloride.

An amount of 2 microliters of the 1 micromolar aptamer solution were dropped onto the surface of the sensors. The sensors with the aptamer solution were then kept in a hydrate container at 80° C. for 10 minutes to allow aptamers to bond to the surface. The excess aptamers were then washed off by dipping in deionized water and the sensors were dried under nitrogen stream. An additional 2 microliters of the aptamer solution was added and washed and dried.

After this, 2 microliters of stock inactivated Zika virus from Zeptomatrix was added to the aptamer coated sensors, and the Zika virus was allowed to bind with the aptamers for 5 minutes. The excess virus was then washed off by dipping in deionized water and drying under nitrogen stream.

The aptamer solution was added to three sample surfaces. The first surface was a polystyrene surface. The second surface was a graphene surface. The third surface was polycarbonate having a well structure and coated with a 50 nm thick layer of gold.

For the polystyrene surface, the aptamer solution wax mixed with stock Zika virus solution, and the mixture was placed on the polystyrene surface. A spacer was placed next to the sample fluid and a cover was placed over the sample fluid and spacer so that the sample fluid had a uniform thickness. In the case of the graphene substrate, the Zika virus solution was added without aptamers. For the gold coated polycarbonate surface, the Zika virus solution was mixed with polystyrene microbeads having a diameter of 0.5 micrometers and coated with aptamers. Aptamers were also immobilized on the gold surface.

For each type of sensor surface, the sample fluid was placed on the sensor surface and the surface was then positioned about 1 mm away from a teraherz horn antenna connected to a network analyzer. The network analyzer was used to measure reflection coefficient (S11) spectra for the sensor surfaces. FIG. 34 shows the reflection coefficient spectra for the polystyrene sensor surface. This graph shows the spectra for the bare substrate, the substrate with attached aptamer, the substrate with Zika and the aptamer, and the substrate with Zika alone. The bare substrate had a minimum reflection coefficient at a frequency of 1.036 THz; the aptamer-coated substrate at 1.066 THz, the Zika-coated substrate at 1.074 THz, and the Zika/aptamer-coated substrate at 1.065 THz. The different minimum reflection coefficient frequencies can allow distinguishing between a sensor having no Zika virus and a sensor where Zika virus is present.

Similar measurements were carried out on the graphene surface. Graphene has zero band gap energy with 0.34 nm thickness, and very high electron mobility of about 10,000 cm²V/s. In some examples, adding aptamers and viruses to a graphene surface can affect the reflection coefficient of the graphene layer due to their electric charge arrangements. The conductivity of graphene can change in unique ways in the presence of some viruses, which can allow graphene to be used as a sensor without aptamers. In this example, the Zika/aptamer complex shifted the reflectance minimum to a lower frequency. The reflectance minimum of bare graphene was at 1.072 THz and the frequency shifted to 1.071 THz when Zika and aptamer were added. However, Zika virus alone shifted the frequency up to 1.085 THz and aptamer alone shifted the frequency to 1.086 THz. The resistance of the graphene was found to change by 26 ohms when Zika virus was added and the resistance changed by 12 ohms when aptamer was added. The increase in resistance can be attributed to a change in the bandgap energy of about 0.8 meV when Zika was deposited and a change of about 1.6 meV when the aptamer was deposited. This appears to demonstrate the teraherz detection of Zika through its field-effect on graphene. FIG. 35 shows the reflection coefficient spectra for the graphene as a bare graphene layer, with aptamer, with Zika virus, and with the Zika/aptamer complex.

Similar measurements were also performed with the sensor including polystyrene microbeads coated with aptamers and gold plated wells on a polycarbonate substrate. The polycarbonate substrate having wells formed therein was first coated with a 50 nm layer of gold by sputtering. The gold layer was functionalized with aptamers. The microbeads functionalized with aptamers were then placed in the wells. The Zika virus solution was then added to the wells. The Zika viruses acted as a bridge, binding to aptamers on the gold surface and the aptamers on the microbeads. Excess microbeads were removed with deionized water. In this example, the reflection coefficient spectrum was measured and then an additional microliter of the Zika virus solution was added and the measurement was performed again. This was repeated several times to see the effect of increasing amounts of Zika virus on the sensor surface. FIG. 36 shows the spectra for the bare gold surface, and the surface after 3 microliters, 4 microliters, 5 microliters, and 6 microliters of Zika solution were added. The frequency of minimum reflection coefficient decreased with each successive addition of Zika virus. The sensitivity was found to be up to 63 Hz per Zika virus. The minimum detectable signal was about 1.6×10⁴ Zika viruses.

In one aspect, the present technology can incorporate nano-meter scale molecules in various devices to achieve a specific functionality without requiring otherwise nanometer scale device gaps or other features. Nanolithograpy is currently used to fabricate devices with nanometer receiving regions to assemble molecules. With this technique one can assemble nanometer scale molecules inside micrometer-scale gaps without requiring expensive nanolithography.

In one aspect, the present technology can use different types of chemically active molecules to assemble specific functional molecules with a layer-by-layer additive technique. The molecules employed as connection and assembly blocks themselves can have specific functionalities enhancing mechanical, thermal, optical, magnetic and electromagnetic functionalities of the over-all device. The method can enable development of very sophisticated nano-devices using convenient glass-ware technologies.

In one aspect, a colorimetric pathogen sensor as disclosed herein can be configured as a whole bacteria colorimetric visual sensor, which can detect bacteria based on their envelope proteins and outer membrane properties without a need for DNA sequencing every time. Such a sensor can use an aptamer specially designed to be selectively attached to specific sites on a given bacteria. The aptamer and bonded bacteria can change the optical, mechanical, thermal, electrical, and/or magnetic properties of the active part of the sensor leading to many changes in addition to color change.

Bacteria are typically large compared to viruses and can be detected using microscopes. These colorimetric sensors enables their detection very inexpensively using visual inspection via unassisted eye. In some aspects, the colorimetric sensors can produce the color change needed for easy detection of bacteria without any need for electricity or a battery.

EXAMPLES

The following examples pertain to further embodiments.

In one example there is provided, a colorimetric pathogen sensor comprising a substrate and an aptamer coupled to the substrate, the aptamer being operable to bind to a target pathogen, wherein, upon the aptamer binding with the target pathogen, reflected light is altered thereby changing apparent color.

In one example of a colorimetric pathogen sensor, the aptamer comprises an oligonucleotide.

In one example of a colorimetric pathogen sensor, the target pathogen comprises at least one of a virus or bacteria.

In one example of a colorimetric pathogen sensor, upon the aptamer binding with the target pathogen, the substrate is caused to bend thereby altering reflected light and changing apparent color.

In one example, a colorimetric pathogen sensor can comprise a base bonded to the substrate by a first bond, wherein the aptamer is bonded to the base by a second bond, the aptamer being operable to bind to the target pathogen by a third bond, and wherein, upon the aptamer binding with the target pathogen, the third bond generates a residual stress on the base that causes the substrate to bend.

In one example of a colorimetric pathogen sensor, the base comprises a coating substantially covering a surface of the substrate.

In one example of a colorimetric pathogen sensor, the base comprises a grating forming parallel strips on a surface of the substrate.

In one example of a colorimetric pathogen sensor, the substrate comprises a beam.

In one example of a colorimetric pathogen sensor, the beam is a cantilever beam.

In one example of a colorimetric pathogen sensor, the cantilever beam is operable to block passage of light prior to the aptamer binding with the target pathogen and to transmit light upon the aptamer binding with the target pathogen and bending of the substrate.

In one example, a colorimetric pathogen sensor can comprise an ink well formed in an ink well substrate and ink disposed in the ink well, wherein the ink is covered by the cantilever beam and upon the aptamer binding with the target pathogen, the cantilever beam bends away from the ink well substrate and releases from the ink from the ink well.

In one example of a colorimetric pathogen sensor, the beam is supported at opposite ends.

In one example of a colorimetric pathogen sensor, the beam comprises a transparent material and the base comprises a dispersive surface on a surface of the beam, and further comprising a reflecting bottom surface disposed below the beam.

In one example of a colorimetric pathogen sensor, the dispersive surface comprises a grating forming parallel strips on a surface of the beam.

In one example, a colorimetric pathogen sensor can comprise a second dispersive surface on the reflecting bottom surface, the dispersive surfaces being operable to reflect different wavelengths of light.

In one example of a colorimetric pathogen sensor, the transparent material comprises silicon nitride.

In one example of a colorimetric pathogen sensor, the substrate comprises a plurality of beams and the substrate is caused to bend by a binding force of the aptamer and the target pathogen moving the target pathogen into a gap between at least two of the plurality of beams.

In one example of a colorimetric pathogen sensor, the substrate comprises at least one of a nitride material or a cellulose material.

In one example of a colorimetric pathogen sensor, the base comprises a metal material.

In one example of a colorimetric pathogen sensor, the metal material comprises at least one of gold, aluminum, or nickel.

In one example, a colorimetric pathogen sensor can comprise a base bonded to the substrate by a first bond, wherein the aptamer is bonded to the base by a second bond, and the aptamer is operable to bind to the target pathogen by a third bond, the second and third bonds being stronger than the first bond, and wherein, upon the aptamer binding with the target pathogen, the third bond generates a residual stress on the base that causes the base to separate from the substrate thereby altering reflected light and changing apparent color.

In one example of a colorimetric pathogen sensor, the second bond generates a second residual stress on the base insufficient to separate the base from the substrate.

In one example of a colorimetric pathogen sensor, the base comprises a plurality of pads arranged on a surface of the substrate.

In one example of a colorimetric pathogen sensor, fewer than all of the plurality of pads are caused to separate from the substrate.

In one example of a colorimetric pathogen sensor, the plurality of pads comprise at least one of a circular configuration, a triangular configuration, or a rectangular configuration.

In one example of a colorimetric pathogen sensor, the base comprises a plurality of microbeads arranged on a surface of the substrate.

In one example, a colorimetric pathogen sensor can comprise an ink well formed in the substrate and ink disposed in the ink well, wherein the ink is covered by the base and upon separation of the base from the substrate the ink is released from the ink well.

In one example of a colorimetric pathogen sensor, the aptamer is bonded to the substrate.

In one example of a colorimetric pathogen sensor, the substrate comprises at least one of microstructures or nanostructures.

In one example of a colorimetric pathogen sensor, the at least one of microstructures or nanostructures comprises at least one of microtextured surfaces or nanotextured surfaces.

In one example, a colorimetric pathogen sensor can comprise a microbead bonded to a second aptamer operable to bind to the target pathogen, wherein the second aptamer binds to the target pathogen and the target pathogen binds to the first aptamer.

In one example of a colorimetric pathogen sensor, a diameter of the microbead is from about 1 μm to about 4 μm.

In one example there is provided, a colorimetric pathogen sensor comprising a plurality of bases and an aptamer bonded to the plurality of bases, the aptamer being operable to bind to a target pathogen, wherein, upon the aptamer binding with the target pathogen, the plurality of bases are grouped together via the bonds to the aptamer, thereby altering reflected light and changing apparent color.

In one example of a colorimetric pathogen sensor, the plurality of bases comprises a plurality of microbeads.

In one example there is provided, a colorimetric pathogen sensor comprising a matrix, an aptamer embedded in the matrix, the aptamer being operable to bind to a target pathogen, and a plurality of particles embedded in the matrix, wherein, upon the aptamer binding with the target pathogen, the matrix expands and changes the position of the plurality of particles relative to one another, thereby altering reflected light and changing apparent color.

In one example of a colorimetric pathogen sensor, the matrix comprises a gel material.

In one example of a colorimetric pathogen sensor, the gel material comprises a hydrogel material.

In one example of a colorimetric pathogen sensor, the matrix comprises a paper material.

In one example of a colorimetric pathogen sensor, the paper material comprises cellulose.

In one example of a colorimetric pathogen sensor, the plurality of particles comprises nanoparticles.

In one example of a colorimetric pathogen sensor, the nanoparticles comprise gold material.

In one example there is provided, a colorimetric pathogen sensor comprising first and second transparent plates, each transparent plate having a dispersive surface, a matrix associated with the first and second plates, and an aptamer embedded in the matrix, the aptamer being operable to bind to a target pathogen, wherein, upon the aptamer binding with the target pathogen, a refractive index of the matrix changes, thereby altering reflected light from the dispersive surfaces and changing apparent color.

In one example of a colorimetric pathogen sensor, the first and second transparent plates are disposed in the matrix and maintained at a fixed distance from one another.

In one example of a colorimetric pathogen sensor, the matrix is disposed between the first and second transparent plates and, upon the aptamer binding with the target pathogen, the matrix expands and changes a distance between the first and second transparent plates, thereby altering reflected light from the dispersive surfaces and changing apparent color.

In one example of a colorimetric pathogen sensor, the dispersive surface comprises a grating forming parallel strips.

In one example of a colorimetric pathogen sensor, the first and second transparent plates comprise silicon nitride material.

The foregoing detailed description describes the invention with reference to specific exemplary embodiments. However, it will be appreciated that various modifications and changes can be made without departing from the scope of the present invention as set forth in the appended claims. The detailed description and accompanying drawings are to be regarded as merely illustrative, rather than as restrictive, and all such modifications or changes, if any, are intended to fall within the scope of the present invention as described and set forth herein. 

1. A colorimetric pathogen sensor, comprising: a substrate; and a molecular recognition group coupled to the substrate, the molecular recognition group being operable to bind to a target pathogen, wherein, upon the molecular recognition group binding with the target pathogen, reflected light is altered thereby changing apparent color.
 2. The colorimetric pathogen sensor of claim 1, wherein the molecular recognition group is at least one of an aptamer, an antigen, and an antibody and wherein the target pathogen comprises at least one of a virus or bacteria.
 3. (canceled)
 4. The colorimetric pathogen sensor of claim 1, wherein, upon the molecular recognition group binding with the target pathogen, the substrate is caused to bend thereby altering reflected light and changing apparent color.
 5. The colorimetric pathogen sensor of claim 4, further comprising a base bonded to the substrate by a first bond, wherein the molecular recognition group is bonded to the base by a second bond, the molecular recognition group being operable to bind to the target pathogen by a third bond, and wherein, upon the molecular recognition group binding with the target pathogen, the third bond generates a residual stress on the base that causes the substrate to bend.
 6. The colorimetric pathogen sensor of claim 5, wherein the base comprises a coating substantially covering a surface of the substrate or wherein the base comprises a grating forming parallel strips on a surface of the substrate.
 7. (canceled)
 8. (canceled)
 9. The colorimetric pathogen sensor of claim 8, wherein the substrate comprises a cantilever beam.
 10. The colorimetric pathogen sensor of claim 9, wherein the cantilever beam is operable to block passage of light prior to the molecular recognition group binding with the target pathogen and to transmit light upon the molecular recognition group binding with the target pathogen and bending of the substrate.
 11. The colorimetric pathogen sensor of claim 9, further comprising an ink well formed in an ink well substrate and ink disposed in the ink well, wherein the ink is covered by the cantilever beam and upon the molecular recognition group binding with the target pathogen, the cantilever beam bends away from the ink well substrate and releases from the ink from the ink well.
 12. The colorimetric pathogen sensor of claim 5, wherein the substrate comprises a beam and the beam is supported at opposite ends.
 13. The colorimetric pathogen sensor of claim 5, wherein the substrate comprises a beam and the beam comprises a transparent material and the base comprises a dispersive surface on a surface of the beam, and further comprising a reflecting bottom surface disposed below the beam.
 14. The colorimetric pathogen sensor of claim 13, wherein the dispersive surface comprises a grating forming parallel strips on a surface of the beam.
 15. The colorimetric pathogen sensor of claim 13, further comprising a second dispersive surface on the reflecting bottom surface, the dispersive surfaces being operable to reflect different wavelengths of light.
 16. (canceled)
 17. The colorimetric pathogen sensor of claim 4, wherein the substrate comprises a plurality of beams and the substrate is caused to bend by a binding force of the molecular recognition group and the target pathogen moving the target pathogen into a gap between at least two of the plurality of beams.
 18. (canceled)
 19. (canceled)
 20. (canceled)
 21. The colorimetric pathogen sensor of claim 1, further comprising a base bonded to the substrate by a first bond, wherein the molecular recognition group is bonded to the base by a second bond, and the molecular recognition group is operable to bind to the target pathogen by a third bond, the second and third bonds being stronger than the first bond, and wherein, upon the molecular recognition group binding with the target pathogen, the third bond generates a residual stress on the base that causes the base to separate from the substrate thereby altering reflected light and changing apparent color.
 22. The colorimetric pathogen sensor of claim 21, wherein the second bond generates a second residual stress on the base insufficient to separate the base from the substrate.
 23. The colorimetric pathogen sensor of claim 21, wherein the base comprises a plurality of pads arranged on a surface of the substrate.
 24. The colorimetric pathogen sensor of claim 23, wherein fewer than all of the plurality of pads are caused to separate from the substrate.
 25. (canceled)
 26. The colorimetric pathogen sensor of claim 21, wherein the base comprises a plurality of microbeads arranged on a surface of the substrate.
 27. The colorimetric pathogen sensor of claim 21, further comprising an ink well formed in the substrate and ink disposed in the ink well, wherein the ink is covered by the base and upon separation of the base from the substrate the ink is released from the ink well.
 28. The colorimetric pathogen sensor of claim 1, wherein the molecular recognition group is bonded to the substrate.
 29. The colorimetric pathogen sensor of claim 28, wherein the substrate comprises at least one of microstructures or nanostructures and wherein the at least one of microstructures or nanostructures comprises at least one of microtextured surfaces or nanotextured surfaces.
 30. (canceled)
 31. The colorimetric pathogen sensor of claim 28, further comprising a microbead bonded to a second molecular recognition group operable to bind to the target pathogen, wherein the second molecular recognition group binds to the target pathogen and the target pathogen binds to the molecular recognition group. 32.-56. (canceled) 